Disruption of cellular calcium homeostasis is a recognized consequence of diabetes-induced hyperglycemia. In certain retinal cell types, elevated intracellular calcium stimulates pathogenic responses that result in retinal leukostasis and vascular hyperpermeability - two vision-threatening hallmarks of non-proliferative diabetic retinopathy (DR). Thus, as a critical intracellular second messenger, Ca2+ may play an important role in high glucose-induced vascular pathology in DR, but the downstream signaling pathways through which pathogenic Ca2+ signals are mediated remain unknown. Recent evidence points to a critical role for Nuclear Factor of Activated T Cells (NFAT) in the pathway linking hyperglycemia-induced Ca2+ flux to these pathologic cellular responses. Elevated intracellular calcium triggers calcineurin (CN)-mediated activation of NFAT, leading to its nuclear translocation. NFAT integrates hyperglycemia-induced calcium flux with other biochemical signals by heterodimeric binding with other transcription factors. These heterodimeric complexes stimulate the transcription of target genes in a manner specific to multiple biochemical cues. For example, in retinal Muller cells, activation of the NFAT pathway leads to the generation of mRNA coding for HIF-1a, COX-2, and VEGF, resulting in elevated retinal VEGF levels, which induces vascular hyperpermeability and retinal edema. Our preliminary data indicate that the majority of high glucose induction of VEGF is CN-dependent. Likewise, in retinal microvascular endothelial cells (RMEC), NFAT transcriptional activity leads to the production of mRNA coding for the inflammatory cytokines CXCL2, VCAM-1, ICAM-1, IL-6, and IL-8, which promote leukostasis and hyperpermeability. Our preliminary data support the links between Ca2+, CN, NFAT, and production and secretion of inflammatory mediators. In order to understand the role of CN/NFAT signaling in DR, its signaling pathways must be characterized in the specific retinal cells involved in the diabetic response: Muller cells produce growth factor and cytokines in response to increases in intracellular calcium, and RMEC both produce and respond to these factors. Using primary cultures of these two cell types, the complicated interactions between hyperglycemia-induced Ca2+ flux, CN activation, NFAT activation, and consequent cell responses will be studied systematically. These studies will lead to the identification of signaling intermediates that may serve as appropriate therapeutic targets. We propose the following studies, aimed at a better understanding of the role of CN/NFAT signaling in retinal vascular disease: 1) comparisons of the effects of high glucose and/or VEGF in the presence of highly selective CN and NFAT inhibitors on cells isolated from mouse or human retinal tissues; 2) comparisons of the relevant effects of high glucose on cells isolated from wild type and NFAT-/- mice or of the effects of high glucose and/or VEGF on NFAT isoform-directed siRNA- treated cells; and 3) determination of the therapeutic efficacy of highly specific CN and NFAT inhibitors in STZ- treated mice. The STZ model has been selected for its specific relevance to non-proliferative DR.